Cardiosphere-derived cells (CDCs) are effective in reducing scar size and regenerating viable myocardium in patients after myocardial infarction (MI), and exert acute cardioprotective effects in animal models of MI. The major benefits of CDCs appear to be mediated by indirect mechanisms. Here we test the overarching hypothesis that the indirect effects of CDCs are mediated by exosomes. Secreted by a wide range of cell types, exosomes are 30-100 nm lipid bilayer vesicles that are enriched in RNAs including microRNAs (miRs). Our preliminary data indicate that exosomes secreted by human CDCs reproduce the therapeutic benefits of CDCs, and are indispensable for the therapeutic benefits of CDCs, in a mouse acute MI model. Injection of CDC-exosomes into the injured heart mimics the structural and functional benefits of CDC transplantation; conversely, inhibition of exosome secretion by CDCs abrogates the therapeutic benefits of transplanted CDCs. Not all exosomes are salutary: Injection of exosomes from dermal fibroblasts, control cells which do not improve post-MI outcomes, had no therapeutic benefit. CDC-exosomes decreased acute cardiomyocyte death and inflammatory cytokine release, while attenuating LV remodeling and fibrosis in the post-MI heart. Preliminary data from microRNA arrays reveal several signature miRs that are highly up-regulated in CDC- exosomes. In contrast, mass spectrometry indicates that the protein composition of CDC-exosomes is conventional and comparable to that of fibroblast-exosomes. Thus, we hypothesize that: i) CDC-exosomes contain a unique complement of miRs that, collectively, mediate many, or all, of the therapeutic effects of CDCs; ii) CDC-exosomes and their constituent miRs favorably modulate apoptosis, inflammation and fibrosis in the post-MI heart; iii) CDC-exosomes improve functional recovery and increase tissue viability post-MI; iv) CDC-exosomes represent a viable cell free therapeutic candidate for cardiac repair. Here we propose to sequence the full RNA content of CDC-exosomes, to determine the molecular basis underlying the therapeutic benefit of CDC-exosomes, and to optimize delivery strategies in vivo. Our ultimate goal is to develop CDC-exosomes as a biologic product to treat MI and HF. Because exosomes are unlike live cells that can migrate actively towards a target, we also seek to test novel delivery methods to target CDC-exosomes selectively to injured myocytes. The proposal is both hypothesis-driven and product-oriented. We seek to answer both mechanistic and translational questions in detail, relying upon state-of-the-art scientific methods and well-established preclinical disease models. CDCs (both autologous and allogeneic) are already in human trials, but cells have their limitations as therapeutic agents. Thus, our proposal, focusing on CDC-exosomes (as cell-free derivatives of CDCs), is of potentially significant translational value. The results will also open up novel insights into the fundamental mechanisms of cell-mediated cardioprotection and cardiac regeneration after acute MI.